Genome-wide identification of CCO gene family in cucumber (Cucumis sativus) and its comparative analysis with A. thaliana

Carotenoid cleavage oxygenase (CCO) is an enzyme capable of converting carotenoids into volatile, aromatic compounds and it plays an important role in the production of two significant plant hormones, i.e., abscisic acid (ABA) and strigolactone (SL). The cucumber plant genome has not been mined for genomewide identification of the CCO gene family. In the present study, we conducted a comprehensive genome-wide analysis to identify and thoroughly examine the CCO gene family within the genomic sequence of Cucumis sativus L. A Total of 10 CCO genes were identified and mostly localized in the cytoplasm and chloroplast. The CCO gene is divided into seven subfamilies i.e. 3 NCED, 3 CCD, and 1 CCD-like (CCDL) subfamily according to phylogenetic analysis. Cis-regulatory elements (CREs) analysis revealed the elements associated with growth and development as well as reactions to phytohormonal, biotic, and abiotic stress conditions. CCOs were involved in a variety of physiological and metabolic processes, according to Gene Ontology annotation. Additionally, 10 CCO genes were regulated by 84 miRNA. The CsCCO genes had substantial purifying selection acting upon them, according to the synteny block. In addition, RNAseq analysis indicated that CsCCO genes were expressed in response to phloem transportation and treatment of chitosan oligosaccharides. CsCCD7 and CsNCED2 showed the highest gene expression in response to the exogenous application of chitosan oligosaccharides to improve cold stress in cucumbers. We also found that these genes CsCCD4a and CsCCDL-a showed the highest expression in different plant organs with respect to phloem content. The cucumber CCO gene family was the subject of the first genome-wide report in this study, which may help us better understand cucumber CCO proteins and lay the groundwork for the gene family's future cloning and functional investigations. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-023-04647-4.


Introduction
Carotenoids, which are naturally occurring isoprenoid compounds, exhibit widespread distribution across various organisms including bacteria, prokaryotes and plants [1,2].Since their discovery in the nineteenth century, more than 700 distinct carotenoids have been identified and characterized [3].In the realm of plants, carotenoids have been found to fulfill diverse roles in essential biological processes [4].They serve as photosynthetic pigments and general antioxidants, facilitating light capture and prevent photo oxidation [5].Throughout the process of carotenoid cleavage into various apocarotenoid products, they assume various functions, including the regulation of plant growth and development and also show response to biotic and abiotic stresses in plants [6].
Plants rely heavily on the Carotenoid Cleavage Oxygenase (CCO) family because they are essential for controlling carotenoid metabolism.The cleavage of carotenoids by CCO enzymes results in the creation of several apocarotenoids, which are important signaling molecules and building blocks for the synthesis of hormones and fragrance chemicals [7].Numerous physiological processes, such as plant development, stress responses, and the regulation of defense systems against diverse environmental challenges, are facilitated by the diverse roles of CCOs.Furthermore, the capacity of CCOs to produce bioactive substances like retinoids and abscisic acid highlights the importance of these entities in regulating plant growth and adaptive responses.The complex role that the CCO family plays in carotenoid metabolism places it at the forefront of influencing the many facets of plant biology and adaptability [8].
Carotenoid cleavage oxygenases (CCOs), which are classified as non-heme iron oxygenases cause the oxidative alteration of carotenoids that yields apocarotenoids.Plants use apocarotenoids as pigments, phytohormones and defensive chemicals [9].Enzymes, which are also known as carotenoid cleavage dioxygenases (CCDs) in some scientific literature, need deoxygenate to begin the oxidative cleavage process [10].In this study, we have opted to refer to them as CCOs for clarity, to distinguish them from the CCD subfamily.
The CCO gene family has been extensively investigated and characterize in numerous plant species, including Oryza sativa [11], Arabidopsis thaliana [12], Solanum Lycopersicum [13] and Capsicum [14].Based on how well their substrate contributes to an epoxy structure, the CCOs can be further separated into nine-cis-epoxide carotenoid dioxygenase (NCED) and carotenoid cleavage dioxygenase (CCDs) [11].
Previous studies have shown the various roles played by genes from the CCD subfamily in plant physiology and development.Aside from these functions, CCD is also involved in photosynthesis, reactions to biotic and abiotic stressors, the production of apocarotenoids, including aromatic volatiles and strigolactones (SLs) and other processes [15].In the case of tomatoes, the LeCCD1 gene is involve in the production of essential flavor volatiles [16].Similar to this, PhCCD1 in petunias is linked to the production of β-ionone, an essential component of the smell present in many plant species Additionally, CCD1 enzymes affect how plants react to stress [17].During leaf senescence, for instance, CCD1 expression increases, suggesting that CCD1 enzymes may be involved in the catalysis of carotenoids and apocarotenoids [18].
Abscisic acid (ABA) is synthesized by the NCED subfamily of genes, which has been thoroughly characterized for this interaction [19].ABA is well-known for its fundamental roles in plant developmental processes and stress signaling [20].9-cis-epoxy carotenoids cleave to create the precursor of ABA known as C15-xanthoxin [21].Interestingly, the first CCO gene discovered, formerly known as Vp14 in maize, is connected to the rate-limiting stage of ABA biosynthesis [22].Likewise, in studies involving Arabidopsis thaliana, it was found that five NCED genes play essential roles in the initial committed step of ABA synthesis [11].For instance, NCED6 and NCED9 were identified as contributors to ABA synthesis during seed development in Arabidopsis thaliana [23].
The precise roles of CCD genes in cucumber remain unclear, while extensive research on CCO genes has been carried out in Arabidopsis thaliana.However, Arabidopsis is not an ideal candidate for investigating CCD enzymes and their functions because it lacks CCDL proteins.Fortunately, CCDL genes have been identified in Citrullus lanatus and Cucumis melo, opening up new possibilities for further exploration [24].Carotenoids are responsible for the vibrant colors in fruits and vegetables, and the CCD enzyme family plays a crucial role in both their production and degradation [25].With the presence of CCDL genes in C. lanatus and C. melo, these species are promising candidates for future research on CCD enzymes and their functions.
Cucumis sativus L., commonly known as cucumber, is a rich source of vitamins, minerals, nutrients, and bioactive compounds.Beyond its role as a staple food, cucumber has a long history of utilization in traditional and contemporary cultures for its therapeutic properties and applications in beauty and skincare.Originating in Asia, cucumbers are now cultivated in both tropical and subtropical regions.Belonging to the Cucurbitaceae family, which comprises 118 genera and 825 species, cucumbers are highly sought after within this plant family due to their exceptional nutritional value [26].
This research undertook a genome-wide exploration of the CCO gene family, examining aspects such as gene structure, domains, motifs, phylogenetic connections, miRNA target locations, and cis-elements.This investigation offers a strong basis for forthcoming studies on the functional roles of CCO genes in cucumbers.It's important to note that cucumber has not previously undergone an exhaustive examination of the CCO gene family.Therefore, delving deeper into the study of CCO genes could yield fresh perspectives for enhancing cucumber's ability to withstand stress.

Sequences retrieval
The NCBI (https:// www.ncbi.nlm.nih.gov) database was used to find the amino acid sequences for the carotenoid cleavage oxygenase (CCO) domain.In order to find CCO genes, these sequences more specifically the RPE65 domain were used in a BLAST-P (Protein-basic local alignment search tool programme) study using the cucurbita genome database (Version 2) (cucurbitgenomics.org/blast).Twelve sequences all were obtained from the cucumber database using this method.The NCBI CDD (Conserved Domain Database) (http:// www.ncbi.nlm.nih.gov/ Struc ture/ cdd/ wrpsb.cgi) was used with preset settings to validate the retrieved amino acid sequences [14].The proteins lacking the conserved domain were then carefully analyzed and deleted.

Determination of physio-chemical properties and subcellular localization of CCO gene
The web programme protparam (https:// web.expasy.org/ protp aram/) was used to find out the length of the carotenoid cleavage oxygenase (CCO) proteins as well as their molecular weight isoelectric point (pI), GRAVY value and instability index.From the cucumber genome database, the gene names, chromosomal locations and protein sequences were retrieved.Additionally, the WoLF PSORT programme (https:// wolfp sort.hgc.jp/) was used to estimate the subcellular localization of the CCO gene [27].

Gene structure, cis regulatory analysis and motif analysis
The gene structure (Intron-Exon) of the CCO genes was displayed using the Gene Structure Display Server (GSDS) (v2.0) (http:// gsds.cbi.pku.edu.cn/) by using genomic and CDS sequence from cucurbita genome database [28].Additionally, the PlantCare database (http:// bioin forma tics.psb.ugent.be/ webto ols/ plant care/ html) was used to find the cis regulatory elements connected to these genes with upstream 1000 bp promoter region from cucurbita genome database (http:// cucur bitge nomics.org/ blast) [29].With a maximum value of 25, motifs were found using the MEME suite programme (http:// meme.sdsc.edu/meme/website/intro.html) and the TBtool was used to display the found motifs.

Phylogenetic analysis
The Molecular Evolutionary Genetic Analysis (MEGA) software was used to carry out the phylogenetic study.The ClustalW method was used to align the amino acid sequences of CCO proteins from Cucumis sativus, Arabidopsis thaliana, Cucurbita pepo, Cucurbita maxima and Oryza sativa.These protein sequences were then used to build a phylogenetic tree using the neighbor-joining (NJ) algorithm, with a bootstrapping value of 1000 replications.The generated tree was displayed using the iTOL website (https:// itol.embl.de/ upload.cgi) [30].

Evolutionary analysis and chromosomal mapping
Ka/Ks ratios were utilized to calculate the CCO genes' divergence time by using TBtool.Default parameters were used, as instructed in the program's instructions.The molecular evolution rate of each gene pair was determined by calculating the ratio of Ka/Ks using paralogous genes.The time of divergence (DT) was calculated by using this T = Ks/2r in which "r" signifies the neutral substiuition rate (6.5*10 -9 ).The gene duplication occurrences were examined using MCScanX v1.0 (Multiple Collinearity Scan toolbox) with the default settings.Dual synteny was performed by using two crops i.e. A. thaliana, C. maxima.The synteny graph was created by using the circus module in TBTool [31].CCO's beginning and ending positions were taken from the Cucumber genome database.Using the TBTool, the chromosomal mapping of the gene was made visible.

GO ontology analysis
The activities of the CCO genes were further validated using GO annotations by gene ontology (GO) term enrichment study.The online programme called ShinyGo v0.741 (http:// bioin forma tics.sdsta te.edu/ go/), was used for better understand of CCO genes function in cucumber.For visualization of biological, molecular and cellular function, an online database shiny Go was used.

Gene expression profiling of phloem content in different parts of cucumber
All CCO genes' expression levels were measured in a range of plant organs, including phloem-rich leaves, pedicles, stalks and fruits, as well as under various biological conditions.Using already collected high through put sequencing data, the expression analysis of the CCO genes were carried out [32].NCBI Gene Expression Omnibus (GEO) used for information on the developmental stages and the reactions of the various organs.

Gene expression profiling of cucumber against cold tolerance
The expression analysis of all CCO genes to enhance cold tolerance in cucumber was performed by applying chitosan oligosaccharide through a spray method.This analysis utilized previously gathered high-throughput sequencing data [33].

Putative miRNA analysis
The cucumber mature miRNA sequence was identified by NCBI geo (https:// www.ncbi.nlm.nih.gov/ geo/).Using psRNATarget, the CDS sequences of all the CCO genes were used to locate the associated micro-RNA (miRNA) sequences.The PsRNA comprehends the function of the CCO genes in cucumber.Shiny Go, an online database was used to visualize biological, molecular, and cellular function.

Identification of CCO gene in cucumis sativus
To identify the CCO gene family in Cucumis sativus, we conducted a search for these genes within the cucumber genome available in the cucurbit genomics database.Mining of cucumber genome identify 4 NCED, 4 CCD and 2 CCDLike genes in CCO gene family.Based on their homologies to A. thaliana in phylogenetic tree, these genes were named.The proteins that encoded by identical gene isoforms and with a truncated RPE65-binding domain were eliminated.The pI, MW, GRAVY, instability index and anticipated subcellular localization of these CsCCO, as well as other physical and chemical details, are shown in Fig. 1 and Table 1.The protein length of CsCCO is in between 91-614 amino acid residues while molecular weight was in range of 10.53 to 67.15KDa.The isoelectric point (pI) values of the identified proteins ranged from 4.72 to 8.78.The GRAVY value shows that all proteis are hydrophilic in nature based on their negative value.In contrast to the remaining proteins, which were determined to be unstable based on the instability index study, CsCCD4a, CsCCD4b, CsCCDL-a, CsCCD7, and CsCCD8 displayed protein stability (as demonstrated by an instability index of less than 40).Predictions of subcellular localization indicated that the majority of CsCCO proteins might have a role in the cytoplasm and chloroplasts.

Phylogenetic analysis of CCO protein
We explored the evolutionary connections among CCO genes in Cucumis sativus by utilizing the MegaX  software.Phylogenetic tree comprising 61 CCO proteins from four distinct species (A.thaliana, C. maxima, C. pepo and oryza sativa) was constructed using maximum-likelihood (ML) method.This was undertaken to enhance our comprehension of the evolutionary connections among CCO proteins.The grouping of phylogenetic tree was based on presence of Arabidopsis gene in each clade.The result showed that 61 CCO proteins divided into seven subfamilies (NCED2, NCED5, NCED6, CCD4, CCD7, CCD8 and CCDLike) (Fig. 2).The results demonstrate that the clade NCED2, CCD7, CCD8 and NCED6 each contains one gene of cucumber.In contrast, the clade encompassing NCED5, CCD4, and CCDLike harbors two genes each.CCDL genes that were retrieved from another crop, its characteristics were not known yet because these were not present in Arabidopsis.Interstingly, O.sativa also containing CCDL genes.

Gene structure, domain and motif analysis
Analysis of exon-intron patterns and conserved motif composition to gain deeper insights into the gene structure characteristics of the CCO gene family, as depicted in Fig. 3. Our investigation of gene structures revealed that the CsCCO genes exhibit a range of exon counts, spanning from 1 to 14 exons.It's noteworthy that the CCD7 subfamily displayed the highest number of both introns and exons.In contrast, the NCED6, NCED2, and CCD8 clades were distinguished by their single-exon structure and the absence of introns.Meanwhile, the CCDlike displayed 9 exons and 7 introns, while the CCD4 exhibited a more complex structure with 13 exons and 11 introns as depicted in Fig. 3. Conserved domain analysis exhibited that all of CsCCO genes contained a RPE65 domain.The motif analysis of CsCCO genes revealed that all members exhibit motifs 2 and 8, with the exception of CCDL-a, which lacks motif 8, and CCDL-b, which lacks motif 2 in Fig. 4.

Synteny analysis and chromosomal mapping
To identify orthologous genes of cucumber in other species, we created a comparative dual synteny of cucumber with Arabidopsis and Cucurbita maxima.Through dual synteny analysis of cucumber with Arabidopsis we found 8 orthologue gene pair (Fig. 6A).The total 14 paralouges gene pair have found in cucumber with cucurbita maxima (Fig. 6B).The comparison of synteny analysis between C. maxima and Arabidopsis revealed a higher number of paralouges genes in C. maxima, suggesting a strong evolutionary connection between them.Moreover, an advanced circos plot representing predicted RNAi-related genes in watermelon demonstrated the existence of paralogous genes within the genome (Fig. 6C).Chromosomal distribution indicated that CsCCO genes are situated across seven chromosomes, as depicted in Fig. 7. Chromosome 6 (Chr6) harbored three CsCCO genes, accounting for 30% of the total.Chromosomes 2, 3, and 4 each contained two CsCCO genes, representing 20.0% for each of them.Chromosome 7 hosted a single CsCCO gene as Fig. 7.

Cis element analysis
Various cis regulatory element were detected in promoter regions of CCO genes using PLANT CARE database, these elements plays pivotal roles in gene transcription initiation.Specific cis-regulatory elements associated with growth, development, phytohormone responses and stress induction are illustrated in Fig. 8.Among these elements, The TGACG motif controls genes involved in defense responses and is linked to jasmonic acid signaling.One fundamental promoter element that is necessary for the start of transcription is the TATA-box.While the ARE is an auxin-responsive element that controls genes linked to growth and development, the CAATbox improves transcription efficiency.Transcription factors for gene regulation are bound by the MYC and MYB motifs; light and stress responses are mediated by G-box; and auxin signaling is linked to growth via AEbox.As-1 is involved in defense mediated by salicylic and jasmonic acids.3-AF1 binding site controls genes in response to ethylene, while auxin signaling is mediated by AuxRR-core.The roles of Box II, chs-CMA1a, Box 4, and Box 4 may vary, and ACE is involved in the control of genes that react to stress and light.ABA-responsive elements called ABRE and ABRE2 control genes in response to abiotic stress.ERE and ethylene are related, and they affect the ripening and senescence of fruit.In salicylic acid-mediated pathogen defense, the TCA element is essential.Gibberellin-related growth genes are regulated by TGA-element and GARE-motif.Salicylic acid signaling involves W box and WUN-motif, auxin-mediated growth involves AE-box, and different hormonal responses require TC-rich repeats.The CGTCA-motif controls the production of jasmonate acid as a defense mechanism against infections and herbivores.These patterns are essential for the expression of certain genes in plants in response to particular hormonal stimuli as in Fig. 8; Supplementary Table S1.
Fig. 4 (A) A phylogenetic tree was constructed to illustrate the relationships among 10 CCO genes found in Cucumis sativus.B To uncover conserved motifs within CCO proteins, an analysis was conducted using the MEME website, and the results were displayed using TBtools.C Domain present in CCO proteins was performed was conducted using NCBI conserved database

GO annotation
Utilizing GO categories, we elucidated the functions of all CCO proteins, encompassing biological processes, molecular functions, and cellular components.Within the biological process category, a significant majority of protein were implicated in carotene catabolic processes (GO:0016119) and terpene catabolic (GO:0016702).Concerning molecular functions, the enriched terms included oxidoreductase activity (GO:0016702).In terms of cellular components, chloroplast stroma (GO:0009570) and plastids (GO:0005575) was predominantly enriched.These findings underscore the diverse roles that these CCO proteins play in cellular metabolism in Fig. 9 and Supplementary Table S3.

Expression analysis of CCO gene for transportation of phloem content in different plant organs in cucumber
We utilized previously published transcriptome data of cucumber to create expression profiles for the predicted CCO genes in response to phloem content in different organs.Phloem-specific transcript profiling was acquired in three separate organs (pedicle, stalk and fruit) using laser microdissection and RNA-Seq technologies.The data showed the expression of CsNCED5a, CsNCED6, CsNCED5b, CsCCD8 but none of CsNCED7 and CsCCDL-b, CsCCD4b and CsNCED2 genes were expressed.CsCCD4a and CsCCDL-a have highest gene expression in all three organ fruit, pedicle and stalk as shown in Fig. 10.

Expression analysis of CCO gene showing improved cold tolerance in cucumber by the treatment of chitosan oligosaccharides
To examine the expression patterns of the predicted CCO genes in cucumber in response to cold stress, we utilized publicly accessible transcriptome data for our analysis.The data was generated to check the pretreated effect of 50mgl −1 chitosan oligosaccharide to cucumber seedlings.For transcriptome analysis, samples of seedlings under cold stress were taken at 0, 3, 12 and 24 h.Distilled water served as the control.The data showed expression of CsNCED6, CsNCED5b, CsNCED5a, CsCCD8, CsCCD7, CsNCED2, CsCCDL-a and CsCCD4b but there is no expression of CsCCDLb and CsCCD4a.CsCCD7 and CsNCED2 gene shows highest expression.The maximum expression shows after 3 h of treatment in CsCCD7 and after 12 h in CsNCED2 but interestingly also show expression after 0 h of treatment.And rest of the gene CsNCED5b, CsNCED5a, CsNCED6, CsCCD4b and CsCCDL-a were down regulated as shown in Fig. 11.

Discussion
In the carotenoid metabolic pathway, carotenoid cleavage oxygenase (CCO) family can oxidize and split carotenoid molecules for production of apocarotenoids.These apocarotenoids serve as essential component in plant hormones, pigments, aromas, flavors and defensive compounds and hold significant biological Fig. 5 The ratio of mutations involving non-synonymous substitutions (Ka) to mutations involving synonymous substitutions (Ks) is shown as Ka/ Ks.On the basis of Ks and Ka values, the gene duplication over selection and evolutionary pressure to paralogous pairings of cucumber CCO genes was determined.The red color indicates the duplication in Million years ago while the blue shows ka, ks and ka/ks ratio importance in the growth and development of plants.Apocarotenoids are linked to hormones such as ABA and SL, as well as non-volatile compounds like crocetin and bixin [34].The CCO gene family is generally present in a wide range of eukaryotic organisms, with a prominent occurrence in a number of plant species.For instance, it has been reported that the CCO genes in Arabidopsis thaliana [35], Malus domestica [15], Vitis vinifera [36], Cucumis melo [37].The CCO genes comprise a unique class of enzymes that can catalyse the Fig. 6 A synteny analysis was conducted on CCO genes in cucumber (Cucumis sativus) using two plant species as references: Arabidopsis thaliana (A) and C. maxima (B).Red and green lines were used to emphasize the syntenic relationships between pairs of CCO genes.These hues can be used to clearly depict the degree of similarity between genetic regions.C Shows the location of CsCCO genes on cucumber chromosomes; lines joining genes on separate chromosomes suggest possible gene duplications production of smaller molecules from the conjugated double-bond structure seen in carotenoids and apocarotenoids.This procedure has a significant impact on how plants grow, develop, produce high-quality crops, and respond to various environmental challenges.Therefore, it is crucial to learn more about the evolutionary relationships and functional characteristics of these CCO genes in plants.
Our investigation involved a comprehensive assessment of the cucumber species' CCO gene family, revealing a relatively small set of 10 CsCCO genes.Through phylogenetic relationship analysis involving A. thaliana, C. pepo, C. maxima and Oryza sativa (as illustrated in Fig. 2), categorized these genes into three primary families: CCD, NCED and CCDLike.These families further consisted of three CCD subfamily members (CsCCD4, CsCCD7 and CsCCD8) and three NCED subfamily members (CsNCED5, CsNCED2, and CsNCED6) and one CCDLike which was consistent to previous scholarly efforts within the field [13], This classification framework builds upon earlier research and contributes to a deeper understanding of the CsCCO gene family's organization and relationships.
Analysis of physicochemical properties revealed that the majority of CsCCO proteins exhibited a length ranging from 91 to 600 amino acids (Table 1), a trend akin to that observed in other plant species [38].Each CsCCO member has unique molecular traits, which are shown in Table 1 after the protein's physicochemical characterization.These differences include things like the GRAVY value, the isoelectric point, and the protein molecular weight.The GRAVY value shows that all proteis are hydrophilic in nature based on their negative value.The RPE65 domain, a distinctive conserved domain within CCO proteins, stands as pivotal to the enzymatic oxidation activity associated with carotenoid cleavage [5].Our examination of conserved domains demonstrated the presence of the RPE65 domain in all CsCCO proteins, displaying a parallel distribution pattern within the same subfamily.All CsCCO proteins shared a common RPE65 domain, which showed a consistent distribution pattern within the same subfamily, according to the study of conserved domains.A striking similarity in distribution patterns was also discovered within the same subfamily when looking at gene structure and motifs (Fig. 4).No conserved motif was present in the all 10 genes.The motif analysis of CsCCO genes revealed that all members exhibit motifs 2 and 8, with the exception of CCDL-a, which lacks motif 8, and CCDL-b, which lacks motif 2 in Fig. 4. Suggesting their significance as pivotal attributes, these motifs may potentially underlie shared functionalities among them.
Multiple earlier investigations, including those conducted previously, have consistently confirmed the localization of the CCO gene on chromosome 7. Analyzing both synteny and dual synteny not only unveiled the Fig. 7 The graphic shows the chromosomal distribution of the cucumber CsCCO gene family.Each of the cucumber chromosomes is represented by the red bars in the picture.The spatial organisation and possible interactions between the CsCCO genes within the cucumber genome are revealed by this investigation.Conversely, the co-regulation or functional links between the CsCCO genes are shown by the green lines.The scale on the map corresponds to the chromosomal distance measured in base pairs (bp).This chromosome map was created using TBtool Fig. 8 A Shows the cis-acting regulatory elements involve in of growth and developmental hormones B Involved in gene expression regulation, light response, defense and drought responsive associated elements of 1000 bp promoter sequences of CCO genes.The intensity defined by the red (highiest) and light red (lowest) during plant biochemical and physiological processes Fig. 9 The CsCCO genes' overlapping functions shown by the Fold Enrichment plot and network.This figure illustrates the processes that CsCCO genes are mostly involved in and gives a brief summary of their functional distribution interconnectivity of CCO genes in various crop species but also highlighted their dispersion across chromosomes 2, 3, 4, 5, 6, 7, and 8.This cumulative research provides a solid groundwork for comprehending the positioning of CCO genes and their broader implications across diverse crop [39].
Mainly the CCO genes were present in cytoplasm and chloroplast [13].As, current study also predict that all ten genes of CCO were predominantly localized in the cytoplasm as shown in (Fig. 1).Although, seven out of ten genes were also present in nucleus and some genes were also present in other organelles as well.Moreover, a parallel inquiry in Saccharum highlighted that specific CCO genes exhibited intricate localization patterns, implying diverse roles across distinct organelles [8].
A multitude of insights regarding the evolutionary background and gene structure can be obtained by closely examining the genomes of different species.Furthermore, these analyses facilitate the transfer of genetic information from a taxonomic group with a high degree of research to one with a lower degree of study [40].The discovery of 14 paralogous genes in CsCCO in this study indicates that genes are replicating via gene duplication.This duplication event provides important new information about gene family expansion, which is frequently seen in the kingdom of plants as a result of tandem and segmental duplications [41].
Gene duplication and differentiation are central mechanisms driving the emergence of novel gene families and functions, with the CCO gene family exemplifying this trend across diverse plant species by target specific genes through complementary base pairing with miR-NAs, allowing them to regulate gene expression by either degrading the miRNA or inhibiting its translation [42].Singh A et.al 2023 [42] miRNA plays an important role in biotic [43] and abiotic stress [44].MicroRNAs (miR-NAs) are essential regulatory molecules that are involved in nearly every biological function, including plant development, growth, and responses to biotic and abiotic stress.They have distinct roles and are highly conserved [45].The study demonstrated that the CsCCOs possesses a wide range of miRNAs with diverse functions, such as concentrating on genes implicated in the dysregulation of stress.Plant response to biotic and abiotic stressors is regulated by csa-miR393b-p3_cme [46].Cucumber yield and size are thought to be regulated by csa-miR393a-p3_cme [46].It is believed that csa-miR408-5p_ptc regulates plant development, growth, and stress response [47].During the process of somatic embryogenesis, csa-miR528-5p_ata controls a number of target mRNAs by either encouraging their degradation, inhibiting their translation, or doing both [48].The findings suggest that miRNAs may have a role in regulating the developmental processes associated with CsCCO genes.
Cis-regulatory elements are crucial for regulating transcriptional levels of gene expression and are often found in the promoter region of genes [49].Studies on cis-regulatory elements showed that motifs like G-box, Box 4, AE-box, and others were present in the largest group, which consisted of 18 elements (58.064%) and have been implicated in metabolism and development.The secondlargest category, consisting of 13 elements (41.935%), was associated with stress response and included motifs such as ABRE, MYB, STRE, and TGACG-motif [49].
The GO enrichment analysis of CsCCO genes indicates that these genes play important roles in pigment synthesis, vitamin A production, and plant defense mechanisms.It also emphasizes the genes' substantial engagement in carotene metabolic and terpene catabolic processes [50].Their potential in carotenoid cleavage reactions is further highlighted by their relationship with carotenoid dioxygenase activity.The significant involvement of CsCCO genes in many metabolic activities inside the chloroplast stroma, especially those pertaining to photosynthesis and pigment production, is indicated by the high enrichment of these genes in this organelle [51].
Consequently, we analyzed the expression profiles of CsCCOs in response to phloem content using quantitative real-time polymerase chain reaction (qRT-PCR) collected fron NCBI geo.Phloem plays a vital role in conveying photosynthetic products and systemic signals between sources and sink organs, crucial for overall plant growth and survival.Cucumber's significance as a model species for studying phloem-related research was emphasized [52].Notably, CsCCD4a and CsCCDL-a exhibited notably high gene expression across fruit, pedicle, and stalk organs, potentially contributing to phloem transcript regulation in different plant parts as in Fig. 10.The high gene expression of CsCCD4a and CsCCDL-a in various plant parts could imply their contribution to regulating phloem-associated transcripts involved in biotic stress defense mechanisms.
Another gene expression from NCBI geo showed the response of CsCCO genes under cold stress.Incorporating chitosan oligosaccharide in the study amplified the cold stress response in cucumber seedlings.RNA sequencing data unveiled the expression profiles of CsCCO gene under these conditions [33].Notably, genes such as CsNCED6, CsNCED5b, CsNCED5a, CsCCD8, CsCCD7, CsNCED2, CsCCDL-a and CsC-CD4b exhibited activation or upregulation in response to cold stress and chitosan oligosaccharide treatment.CsNCED2 and CsCCD7 showed highest gene expression.This sheds light on their potential roles in the plant's defense and adaptability mechanisms under adverse conditions.
The results of this study offer new perspectives on the functional diversity and evolutionary aspects of the CCO gene family in plants.These insights will prove valuable for future research, aiding in the exploration and cloning of these genes.The thorough genome-wide identification and characterization conducted in this study pave the way for additional investigations in this field.

Conclusion
In conclusion, this comprehensive genome-wide analysis has shed light on the previously unexplored CCO gene family in cucumber (Cucumis sativus L.).We have identified 10 distinct CCO genes, classified into three subfamilies (NCED, CCD and CCDLike) and elucidated their structural, functional, and regulatory properties.The presence of cis-elements associated with growth, development, and stress responses highlights the multifaceted roles of CCO genes in cucumber.Moreover, miRNA regulation, purifying selection, and differential expression patterns in response to chitosan oligosaccharides and phloem content provide valuable insights into the functional diversity of CsCCO genes.This pioneering study paves the way for future investigations and a deeper understanding of cucumber CCO proteins, their roles and potential applications in improving cucumber's response to environmental challenges.

Fig. 1
Fig.1Heat Map illustrating the sub-cellular localization of all CsCCO genes to the nucleus, cytoplasm, chloroplast, golgi apparatus, mitochondria, plasmid, peroxisomes of the plant cell.Blue color symbolizes the absence of the relevant gene in the indicated region, grey color predict the minimal functional presence of the relevant gene and red color shows the maximum functional importance of the relevant gene in the indicated region

Fig. 2 A
Fig.2A phylogenetic tree depicting the relationships among CCO proteins in four plant species Cucumis sativus (Cs), Arabidopsis thaliana (At), Cucurbita pepo (Cp),Cucurbita maxima (Cm) and Oryza sativa was constructed using the ClustalW program in MEGA-X.The neighbor-joining (NJ) method was employed for tree construction, with a bootstrap repeat value of 1000 iterations.The phylogenetic tree was visualized using the iTOL online platform.CCO proteins from C. sativus are highlighted by White Box, while CCO proteins from A. thaliana, C. maima, C. pepo and Oryza sativa are depicted in black characters

Fig. 3
Fig. 3 The gene structure of CCO genes, revealing the count of introns and exons.Yellow lines indicate the exons, plane line indicate the introns while blue line indicate upstream and downstream

Fig. 10
Fig. 10 Heatmap showing phloem content in different plant organs.The red color shows the highest gene expression and skin color shows medium expression as well as blue color show lowest gene expression

Fig. 11
Fig. 11 Heatmap showing expression of cucumber seedlings after exposure of chitosan oligosaccharide.The color red signifies highest gene expression, while grey represents a down regulation of gene expression.Blue indicates the null expression.Additionally, the legend provides information about the scale of expression values

Table 1
Non-redundant genes found in the cucumber genome include information on the NCED, CCD, and CCDLike genes in the CCO gene family.Abbreviation: AA: Amino acid, Mw: Molecular weight, pI: Isoelectric poin